Novel CD40:CD154 binding interruptor compounds and use thereof to treat immunological complications

ABSTRACT

The present invention relates to novel CD40:CD154 binding interrupter compounds and use of these compounds and pharmaceutical compositions comprising them, to treat conditions associated with inappropriate CD154 activation in a subject. Specifically, this invention provides compounds which are identified by screening a library of small molecules for those that are capable of specifically binding CD154 and interrupting CD40:CD154 interaction.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel CD40:CD154 binding interruptercompounds and use of these compounds and pharmaceutical compositionscomprising them, to treat conditions associated with inappropriate CD154activation in a subject. Specifically, this invention provides compoundswhich are identified by screening a library of small molecules for thosethat are capable of specifically binding CD154 and interruptingCD40:CD154 interaction.

BACKGROUND OF THE INVENTION

Data establishing that T cell activation requires both T cell receptor(“TCR”) mediated signals and simultaneously delivered costimulatorysignals have accumulated over the past twenty years. For example,antibody production by B lymphocytes in response to protein antigensrequires a specific, costimulatory interaction with T lymphocytes. ThisB cell/T cell interaction is mediated through several receptor-ligandbinding events in addition to engagement of the TCR. See, e.g., Noelleet al. Immunology Today 13: 431-433 (1992). See also Hollenbaugh et al.EMBO J. 11: 4313-4321 (1992). These additional binding events includethe binding of CD40 on B cells to CD154 (CD40L, and also known as gp39,T-BAM, 5c8 antigen, CD40CR and TRAP) on T cells. Human CD40 is a 50kilodalton cell surface protein expressed on mature B cells, as well asmacrophages, dendritic cells, fibroblasts and activated endothelialcells. CD40 belongs to a class of receptors involved in cell signallingand in programmed cell death, including Fas/CD95 and the tumor necrosisfactor (TNF) alpha receptor. Human CD154, a 32 kD type II membraneglycoprotein having homology to TNF alpha, is a member of the TNF familyof receptors and is transiently expressed primarily on activated Tcells. CD40:CD154 binding has been shown to be required for Tcell-dependent antibody responses. In particular, CD40:CD154 bindingprovides anti-apoptotic and/or lymphokine stimulatory signals. See,e.g., Karpusas et al. Structure 15, 1021-1039 (1995), U.S. patentapplication Ser. No. 09/180,209 and WO97/00895, the disclosures of allof which are hereby incorporated by reference.

The importance of CD40:CD154 binding in promoting T cell dependentbiological responses is underscored by the development of X-linkedhyper-IgM syndrome (X-HIGM) in humans lacking functional CD154. Theseindividuals have normal or high IgM levels, but fail to produce IgG, IgAor IgE antibodies. Affected individuals suffer from recurrent, sometimessevere, bacterial infection (most commonly Streptococcus pneumoniae,Pneumocystis carinii and Hemophilus influenzae) and certain unusualparasitic infections, as well as an increased incidence of lymphomas andabdominal cancers. These clinical manifestations of disease can bemanaged through intravenous immunoglobulin replacement therapy.

The effects of X-HIGM are simulated in animals rendered nullizygous forthe gene encoding CD154 (knockout animals). Studies with nullizygoteshave confirmed that, while B cells can produce IgM in the absence ofCD40:CD154 binding, they are unable to undergo isotype switching, or tosurvive normally and undergo affinity maturation. In the absence of afunctional CD40:CD154 interaction, spleen and lymph node germinalcenters do not develop properly, and the development of memory B cellsis impaired. These defects contribute to a severe reduction or absenceof a secondary (mature) antibody response.

Individuals with X-HIGM and CD154 nullizygotes also have defects incellular immunity. These defects are manifested by an increasedincidence of Pneumocystis carinii, Histoplasma capsulatum, Cryotococcusneoformans infection, as well as chronic Giardia lambli infection.Murine nullizygotes are deficient in their ability to fight Leishmaniainfection. Many of these cell-mediated defects are reversible byadministration of IL-12 or IFN-gamma. These data substantiate the viewthat CD40:CD154 binding promotes the development of Type I T-helper cellresponses. Further support is derived from the observation thatmacrophage activation is defective in CD154-deficient settings, and thatadministration of anti-CD154 antibodies to mice diminished their abilityto clear Pneumocystis infection. Blockade of CD40:CD154 binding appearsto reduce the ability of macrophages to produce nitric oxide, whichmediates many of the macrophages' pro-inflammatory activities. It shouldbe noted, however, that mammals (including humans) who lack functionalCD154 do not develop significant incidences of viral infection.

A number of preclinical studies, including those described inco-pending, commonly assigned PCT patent applications published asWO98/30241, WO98/30240, WO98/52606, WO98/58669 and WO99/45958, describethe promise of agents capable of interrupting CD40:CD154 binding asimmunomodulating agents. In murine systems, antibodies to CD154 blockprimary and secondary immune responses to exogenous antigens, both invitro and in vivo. Antibodies to CD154 cause a reduction in germinalcenters in mice and monkeys, consistent with data on CD154immunodeficiency. Administration of three doses of anti-CD154 antibodyto lupus-prone mice, age three months, substantially reduced titersagainst double-stranded DNA and nucleosomes, delayed the development ofsevere nephritis, and reduced mortality. Moreover, administration ofanti-CD154 antibodies to mice age five to seven months with severenephritis was shown to stabilize or even reverse renal disease.Anti-CD154 antibodies given concomitantly with small resting allogeneiclymphocytes permitted unlimited survival of mouse pancreatic isletallografts. In other animal models, interference with CD40:CD154 bindinghas been demonstrated to reduce symptoms of autoimmune disease (e.g.,multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease),graft rejection (e.g., cardiac allograft, graft-versus-host disease),and mercuric chloride induced glomerulonephritis, which is mediated byboth humoral and cellular mechanisms.

Such studies with anti-CD154 antibodies demonstrate the role of CD154 asa critical target for modulating immune responses.

These studies establish the utility of CD40:CD154 binding interruptersas therapeutic agents. As a result, they also suggest the potential ofnovel CD40:CD154 binding interrupters.

SUMMARY OF THE INVENTION

The present invention provides novel compounds, other than anti-CD154antibodies and soluble CD40 or CD40 fusion proteins, that specificallybind CD154 and interrupt CD40:CD154 interaction. This invention alsoprovides pharmaceutical compositions comprising these compounds. Theinvention also provides methods using these compounds to treatconditions associated with inappropriate CD154 activation.

The foregoing and other objects, features and advantages of the presentinvention, as well as the invention itself, will be more fullyunderstood from the following description of preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

The following discussion illustrates and exemplifies the variety ofcontexts and circumstances in which the invention can be practiced, aswell as providing specific embodiments of the invention.

Novel CD40:CD154 Binding Interruptors

In a preferred embodiment, this invention provides a generic compound,which binds to CD154 and preferably interrupts CD40:CD154 interaction,represented by the formula (Formula I):

wherein:

-   R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from    the group consisting of H, C1-5 alkyl, C1-5 alkyl-aryl, C1-5    alkyl-cycloalkyl, C1-5 alkyl-heteroaryl, C1-5 alkenyl-heterocyclo,    cycloalkyl, cycloalkyl-aryl, C1-5 alkenyl-aryl, CR^(g)R^(h)CO₂H and    CR^(g)R^(h)CO₂alkyl; wherein aryl or heteroaryl are optionally    substituted with one to four substituents selected from R^(d) and    alkyl is optionally substituted with R^(i); or R¹ and R², R³ and R⁴,    R⁵ and R⁶, and R⁷ and R⁸ independently form, together with the    nitrogen to which they are attached, a 5-6 membered ring which is    optionally substituted with a heteroatom selected from the group    consisting of nitrogen, sulfur and oxygen, wherein the nitrogen may    optionally be substituted with alkyl or alkyl-aryl and the ring may    optionally be substituted with R^(j);-   R^(a), R^(b) and R^(c) are independently selected from the group    consisting of H, alkyl and alkyl-aryl;-   R^(d) is selected from the group consisting of halogen, alkyloxy,    NO₂, NH₂, alkyl, SO₂NH₂, hydroxyl and aryl;-   R^(e) and R^(f) are independently selected from the group consisting    of H, alkyl and cycloalkyl; or R^(e)R^(f) together with the atom to    which they are attached form a 4-7 membered ring containing zero to    three heteroatoms selected from the group consisting of nitrogen,    oxygen and sulfur, wherein R^(e)R^(f) together with the atom to    which they are attached may be fused to one or two aromatic rings;-   R^(g) and R^(h) are independently selected from the group consisting    of H, alkyl, aryl, cycloalkyl, alkyl-aryl and alkyl-cycloalkyl;    wherein alkyl is optionally substituted with R^(k); and aryl is    optionally substituted with R^(l); or R^(g)R^(h) together with the    carbon to which they are attached form a 5-8 membered ring which may    be bicyclic;-   R^(i) is selected from the group consisting of: NR^(e)R^(f),    alkyloxy, aryloxy, C(O)aryl, aryl, OC(O)alkyl, alkylaryloxy and    hydroxyl; wherein alkyl is optionally substituted with halogen and    aryl is optionally substituted with R^(d);-   R^(j) is selected from the group consisting of (CH₂)_(y)NR^(e)R^(f),    (CH₂)_(z)OH, CO₂H, C(O)N(alkyl)₂, C(O)NH₂, alkylaryl and aryl;-   wherein aryl is optionally substituted with one to four substituents    selected from R^(d);-   R^(k) is selected from the group consisting of C(O)NH₂, C(O)OH,    alkylthio, NH₂, heteroaryl, heteroalkyl, NHC(NH)NH₂, C1-5    alkylhydroxyl, hydroxyl and alkyloxy;-   R¹ is selected from the group consisting of hydroxyl, NO₂ and C1-5    alkyl;-   U is selected from the group consisting of O, S(O)_(y), NR^(c),    NR^(c)C(O), NR^(c)CO(O)NR^(c);-   W is selected from the group consisting of (CR^(a)R^(b)),    (CR^(a)R^(b))_(y)U(CR^(a)R^(b))_(y), C2-3 alkynyl, C2-3 alkenyl;-   each X is independently 0-3;-   each Y is independently 0-2;-   each Z is independently 1-2.

“Alkyl”, as well as other groups having the prefix “alk”, such asalkoxy, alkanoyl, means carbon chains which may be linear or branched orcombinations thereof. Examples of alkyl groups include methyl, ethyl,propyl, isopropyl, butyl, sec and tert-butyl, pentyl, hexyl, heptyl,octyl, nonyl, and the like.

“Alkenyl” means carbon chains which contain at least one carbon-carbondouble bond, and which may be linear or branched or combinationsthereof. Examples of alkenyl include vinyl, allyl, isopropenyl,pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl,and the like.

“Alkynyl” means carbon chains which contain at least one carbon-carbontriple bond, and which may be linear or branched or combinationsthereof. Examples of alkynyl include ethynyl, propargyl,3-methyl-1-pentenyl, 2-heptynyl and the like. “Cycloalkyl” means mono-or bicyclic saturated carbocyclic rings, each of which have from 3 to 10carbon atoms. The term also includes monocyclic rings fused to an arylgroup in which the point of attachment is on the non-aromatic portion.Examples of cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and thelike.

“Aryl” means mono- or bicyclic aromatic rings containing only carbonatoms. The term also includes an aryl group fused to a monocycliccycloalkyl or monocyclic heterocyclyl group in which the point ofattachment is on the aromatic portion. Examples of an aryl group includephenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3dihydrobenzofuranyl, benzopyranyl, 1,4-benzodioxanyl, and the like.

“Heteroaryl” means a mono- or bicyclic aromatic ring containing at leastone heteroatom selected from N, O and S, with each ring containing 5 to6 atoms. Examples of heteroaryl include pyrrolyl, isoxazolyl,isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl, thiadiazolyl,thiazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl, triazinyl,thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl,benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl,furo(2,3-b)pyridyl, quinolyl, indolyl, isoquinolyl, and the like.

“Heterocyclyl” means mono- or bicyclic saturated rings containing atleast one heteroatom selected from N, S and O, each of said ring havingfrom 3 to 10 atoms in which the point of attachment may be carbon ornitrogen. The term also includes monocyclic heterocycle fused to an arylor heteroaryl group in which the point of attachment is on thenon-aromatic portion. Examples of “heterocyclyl” include pyrrolidinyl,piperidinyl, piperazinyl, imidazolidinyl, 2,3-dihydrofuro(2,3-b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl,tetrahydroisoquinolinyl, dihydroindolyl, and the like. The term alsoincludes partially unsaturated monocyclic rings that are not aromatic,such as 2- or 4 pyridones attached through the nitrogen orN-substituted-(1H,3H) pyrimidine-2,4-diones (N-substituted uracils).

“Halogen” includes fluorine, chlorine, bromine and iodine.

In a more preferred embodiment, this invention provides a compound thatis a subgenera of the compound of Formula (I), said compound, whichbinds to CD154 and preferrably interrupts CD40:CD154 interaction, beingrepresented by the formula (Formula II):

wherein:

-   R⁹ is selected from the group consisting of C1-5 alkyl-aryl,    cycloalkyl and alkenylaryl; wherein the alkyl is optionally    substituted with aryl and the aryl is optionally substituted with    one to four halogens, aryl, NH₂ or NO₂;-   R¹⁰ is selected from the group consisting of alkyl and cycloalkyl;    and-   R¹¹ is selected from the group consisting of H and alkyl.

In another more preferred embodiment, this invention provides a compoundthat is another subgenera of the compound of Formula (I) and is asubgenera of the compound of Formula (II), said compound, which binds toCD154 and preferably interrupts CD40:CD154 interaction, beingrepresented by the formula (Formula III):

wherein:

-   R⁹ is selected from the group consisting of C1-5 alkyl-aryl; wherein    alkyl and aryl are optionally substituted with aryl.

This invention also provides all stereoisomers, including enantiomers,of each of the compounds above. In a preferred embodiment, the compoundsare derived from S-amines (L amino acids).

The most preferred embodiments of this invention are the twenty-onecompounds shown below. All twenty-one compounds are compounds accordingto Formula (I). All twenty-one compounds are derived from S-amines.Seventeen of the twenty-one compounds are also compounds according toFormula (II); and five of these seventeen compounds are also compoundsaccording to Formula (III).

Compound 1, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II); compound 1 being represented by the formula:

wherein: compound 1 is derived from S-amines.

Compound 2, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (I); compound 2 being represented by the formula:

wherein: compound 2 is derived from S-amines.

Compound 3, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II); compound 3 being represented by the formula:

wherein: compound 3 is derived from S-amines.

Compound 4, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (III); compound 4 being represented by the formula:

wherein: compound 4 is derived from S-amines.

Compound 5, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (III); compound 5 being represented by the formula:

wherein: compound 5 is derived from S-amines.

Compound 6, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (III); compound 6 being represented by the formula:

wherein: compound 6 is derived from S-amines.

Compound 7, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II); compound 7 being represented by the formula:

wherein: compound 7 is derived from S-amines.

Compound 8, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II); compound 8 being represented by the formula:

wherein: compound 8 is derived from S-amines.

Compound 9, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II);

compound 9 being represented by the formula: wherein: compound 9 isderived from S-amines.

Compound 10, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II); compound 10 being represented by the formula:

wherein; compound 10 is derived from S-amines.

Compound 11, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II), compound 11 being represented by the formula:

wherein: compound 11 is derived from S-amines.

Compound 12, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II); compound 12 being represented by the formula:

wherein: compound 12 is derived from S-amines.

Compound 13, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (III); compound 13 being represented by theformula:

wherein: compound 13 is derived from S-amines.

Compound 14, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II), compound 14 being represented by the formula:

wherein: compound 14 is derived from S-amines.

Compound 15, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (III); compound 15 being represented by theformula:

wherein: compound 15 is derived from S-amines.

Compound 16, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II), compound 16 being represented by the formula:

wherein: compound 16 is derived from S-amines.

Compound 17, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (I), compound 17 being represented by the formula:

wherein: compound 17 is derived from S-amines.

Compound 18, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (I), compound 18 being represented by the formula:

wherein: compound 18 is derived from S-amines.

Compound 19, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (I), compound 19 being represented by the formula:

wherein: compound 19 is derived from S-amines.

Compound 20, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II), compound 20 being represented by the formula:

wherein: compound 20 is derived from S-amines.

Compound 21, which interrupts CD40:CD154 interaction, is a compoundaccording to Formula (II), compound 21 being represented by the formula:

wherein: compound 21 is derived from S-amines.

In a preferred embodiment, this invention provides a compound of Formula(I), (II) or (III), wherein said compound binds CD154 specifically, andpreferably interrupts CD40:CD154 interaction. Preferably these compoundshave an IC₅₀ for CD40:CD154 interruption of less than or equal toapproximately 310 μM; more preferably less than or equal toapproximately 125 μM; and even more preferably less than or equal toapproximately 50 μM.

In a more preferred embodiment, the invention provides a compound ofFormula (I), (II) or (III), wherein said compound binds CD154specifically; is a CD40:CD154 binding interrupter, and is not aTNF-α:TNFRp60 binding interrupter (i.e., does not bind to TNF-α and isnot a TNF-α:TNFRp60 binding interrupter). Preferably, these compoundshave an IC₅₀ for CD40:CD154 binding interruption of less thanapproximately 125 μM and an IC₅₀ for TNF-α:TNFRp60 of more 125 μM.

IC₅₀, as used herein, is the concentration at which a compoundinterrupts the interaction of a protein and its receptor to 50%interaction in the assay shown in Example 7 (for interruption ofCD40:CD154 interaction) and Example 8 (for interruption of TNF-α:TNFRp60interaction). Examples of such protein and receptor pairs are CD40:CD154and TNF-α:TNFRp60.

Compounds 4-6 and 11 were shown to have IC₅₀ for CD40:CD154 bindinginterruption in the A range (less than 50 μM). Compounds 1, 7-10 and 13were shown to have IC₅₀ for CD40:CD154 binding interruption in the Brange (50 μM to 125 μM). Compounds 2-3 were shown to have IC₅₀ forCD40:CD154 binding interruption in the C range (greater than 125 μM). Inaddition, compounds 12, 14 and 16-21 are considered to have IC₅₀ forCD40:CD154 binding interruption in the B range (50 μM to 125 μM), andcompound 15 in the A range (less than 50 μM). Compounds 1-4 were shownto have IC₅₀ of greater than 125 μM for TNF-α:TNFRp60 bindinginterruption. Table I lists the IC₅₀ for CD40:CD154 binding interruptionand the mass spectrometry data for these twenty-one compounds. TABLE ICompound IC50 Mass Spec * ** 1 B 443.16 ((M + 2)/2) 2 C 434.22 ((M +2)/2) 3 C 434.22 ((M + 2)/2) 4 A 861.4 (M + 1) 5 A 469.3 ((M + 2)/2) 6 A455.23 ((M + 2)/2) 7 B 906.45 (M + 1) 8 B 876.43 (M + 1) 9 B 876 (M + 1)10 B 953 (M + 1) 11 A 982.24 (M + 1) 12 B 430.42 ((M + 2)/2) 13 B 442.24((M + 2)/2) 14 B 470.22 ((M + 2)/2) 15 A 438.25 ((M + 2)/2) 16 B 430.29((M + 2)/2) 17 B 417 ((M + 2)/2) 18 B 485 ((M + 2)/2) 19 B 457 ((M +2)/2) 20 B 438.27 ((M + 2)/2) 21 B 418.24 ((M + 2)/2)* M is for molecular mass.** The formulas in the parenthesis show what the numbers represent.

The chemical syntheses of compounds 4, 7-11 are shown in Examples 1-6. Aperson skilled in the art would appreciate that the other compounds(compounds 1-3, 5-6 and 12-21) may be synthesized from intermediatesand/or products disclosed in Examples 1-6 by routine methods. Theskilled artisan would also appreciate that other routine methods may beused to synthesize these compounds. It should be understood that aperson skilled in the art can synthesize any compound of Formula (I),(II) or (III) using conventional methods.

A specific assay to determine whether a compound interrupts CD40:CD154interaction is shown in Example 7. A specific assay to determine whethera compound interrupts TNF-α:TNFRp60 interaction is shown in Example 8.Other well-known assays may be used for both determinations. One suchassay is shown to determine whether a compound interrupts CD40:CD154interruption is shown in Example 9. The interaction of CD40 and CD154results in up-regulation of I-CAM1. A CD40:CD154 binding interrupterinhibits that up-regulation.

Conditions Associated with Inappropriate CD154 Induced Activation in aSubject

The CD40:CD154 binding interrupter compounds provided by this inventioncan be used to prevent or treat subjects having conditions associatedwith inappropriate CD154 induced activation. Treating a conditionassociated with inappropriate CD154 induced activation in a subjectincludes, inter alia, attenuating severity of the condition, suppressingeffects of the condition, inhibiting the condition and reversing thecondition.

Examples of conditions associated with inappropriate CD154 mediatedactivation in a subject, include, inter alia: an unwanted immuneresponse, an unwanted inflammatory response, an autoimmune disease, anallergy, an inhibitor response to a therapeutic agent, rejection of adonor organ and a B cell cancer.

Examples of conditions associated with inappropriate CD154 mediatedactivation in a subject, include, inter alia: systemic lupuserythematosis, lupus nephritis, lupus neuritis, asthma, chronicobstructive pulmonary disease, bronchitis, emphysema, multiplesclerosis, uveitis, Alzheimer's disease, traumatic spinal cord injury,stroke, atherosclerosis, coronary restenosis, ischemic congestive heartfailure, cirrhosis, hepatitis C, diabetic nephropathy,glomerulonephritis, osteoarthritis, rheumatoid arthritis, psoriasis,atopic dermatitis, systemic sclerosis, radiation-induced fibrosis,Crohn's disease, ulcerative colitis, multiple myeloma and cachexia.

Subjects

The CD40:CD154 binding interrupter compounds provided by this inventioncan be administered for treatment or prophylaxis to any mammaliansubject suffering from or about to suffer a condition associated withinappropriate CD154 activation. Preferably, the subject is a primate,more preferably a higher primate, most preferably a human. In otherembodiments, the subject may be a mammal of commercial importance, or acompanion animal or other animal of value, such as a member of anendangered species. Thus, a subject may be, inter alia, sheep, horses,cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters,gerbils, rats and mice.

Route of Administration

The CD40:CD154 binding interrupter compounds provided by this inventionmay be administered in any manner which is medically acceptable.Depending on the specific circumstances, local or systemicadministration may be desirable. Local administration may be, forexample, by subconjunctival administration. Preferably, the interruptercompound is administered via an oral, an enteral, or a parenteral routesuch as by an intravenous, intraarterial, subcutaneous, intramuscular,intraorbital, intraventricular, intraperitoneal, subcapsular,intracranial, intraspinal, topical or intranasal injection, infusion orinhalation. The interrupter compound also may be administered byimplantation of an infusion pump, or a biocompatible or bioerodiablesustained release implant, into the subject.

Dosages and Frequency of Treatment

Generally, the methods described herein involve administration of theCD40:CD154 binding interrupter compounds at desired intervals (e.g.,daily, twice weekly, weekly, biweekly, monthly or at other intervals asdeemed appropriate) over at least a two- or three-week period. Theadministration schedule is adjusted as needed to treat the conditionassociated with inappropriate CD154 activation in the subject. Aparticular treatment regime can be repeated, in the event of asubsequent episode of illness.

The amount and frequency of dosing for any particular CD40:CD154 bindinginterrupter compound to be administered to a patient for a givenimmunological disease associated with inappropriate CD154 inducedactivation is within the skills and clinical judgement of ordinarypractitioners of the medical and pharmaceutical arts. The general dosageand administration regime may be established by preclinical and clinicaltrials, which involve extensive but routine studies to determine theoptimal administration parameters of the compound. Even after suchrecommendations are made, the practitioner will often vary these dosagesfor different subjects based on a variety of considerations, such as theindividual's age, medical status, weight, sex, and concurrent treatmentwith other pharmaceuticals. Determining the optimal dosage andadministration regime for each synthetic CD40:CD154 binding interruptercompound used is a routine matter for those of skill in the medical andpharmaceutical arts.

Generally, the frequency of dosing may be determined by an attendingphysician or similarly skilled practitioner, and might include periodsof greater dosing frequency, such as at daily or weekly intervals,alternating with periods of less frequent dosing, such as at monthly orlonger intervals.

To exemplify dosing considerations for a CD40:CD154 binding interrupter,the following examples of administration strategies are given for ananti-CD154 mAb. Such illustrative dosing amounts could easily beadjusted for other types of anti-CD154 compounds according to thepresent invention. In general, single dosages of between about 0.05 andabout 50 mg/kg patient body weight are contemplated, with dosages mostfrequently in the 1-20 mg/kg range. For acute treatment, such as beforeor at the time of transplantation, or in response to any evidence thatgraft rejection is beginning, an effective dose of a representativeantibody (such as mAb 5c8), which may serve as a guideline for thedosages of a compound according to this invention, ranges from about 1mg/kg body weight to about 20 mg/kg body weight, administered daily fora period of about 1 to 5 days, preferably by bolus intravenousadministration. The same dosage and dosing schedule may be used in theload phase of a load-maintenance regimen, with the maintenance phaseinvolving intravenous or intramuscular administration being patterned onthat of an anti-CD154 mAb in a range of about 0.1 mg/kg body weight toabout 20 mg/kg body weight, for a treatment period of anywhere fromweekly to 3 month intervals. Chronic treatment may also be carried outby a maintenance regimen, patterned on that in which antibodies areadministered by intravenous or intramuscular route, in a range of about0.1 mg/kg body weight to about 20 mg/kg body weight, with interdoseintervals ranging from about 1 week to about 3 months. In addition,chronic treatment may be effected by an intermittent bolus intravenousregimen, patterned on that in which between about 1.0 mg/kg body weightand about 100 mg/kg body weight of an anti-CD154 mAb are administered,with the interval between successive treatments being from 1 to 6months. For all except the intermittent bolus regimen, administrationmay also be by oral, pulmonary, nasal or subcutaneous routes.

For treatment, a CD40:CD154 binding interrupter compound can beformulated in a pharmaceutical or prophylactic composition whichincludes, respectively, a pharmaceutically or prophylactically effectiveamount of the CD40:CD154 binding interrupter compound dispersed in apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical or prophylactic composition can also include apharmaceutically or prophylactically effective amount of anotherimmunosuppressive or immunomodulatory compound, including withoutlimitation: an agent that interrupts T cell costimulatory signaling viaCD28 (e.g., CTLA4-Ig), CD80 or CD86; an agent that interruptscalcineurin signaling (e.g., cyclosporin, a macrolide such tacrolimus,formerly known as FK506); a corticosteroid; or an antiproliferativeagent (e.g., azathioprine). Other therapeutically effective compoundssuitable for use with the CD40:CD154 binding interrupter includerapamycin (also known as sirolimus); mycophenolate mofetil (MMF),mizoribine, deoxyspergualin, brequinar sodium, leflunomide, azaspiraneand the like.

Combination therapies according to this invention for treatment of acondition associated with inappropriate CD154 activation in a subjectinclude the use of a synthetic CD40:CD154 binding interruptor compoundtogether with agents targeted at B cells, such as anti-CD19, anti-CD28or anti-CD20 antibody (unconjugated or radiolabeled), IL-14 antagonists,LJP394 (LaJolla Pharmaceuticals receptor blocker), IR-1116 (Takeda smallmolecule) and anti-Ig idiotype monoclonal antibodies. Alternatively, thecombinations may include T cell/B cell targeted agents, such as CTLA4Ig,IL-2 antagonists, IL-4 antagonists, IL-6 antagonists, receptorantagonists, anti-CD80/CD86 monoclonal antibodies, TNF, LFA1/ICAMantagonists, VLA4/VCAM antagonists, brequinar and IL-2 toxin conjugates(e.g., DAB), prednisone, anti-CD3 mAb (OKT3), mycophenolate mofetil(MMF), cyclophosphamide, and other immunosuppressants such ascalcineurin signal blockers, including without limitation, tacrolimus(FK506). Combinations may also include T cell targeted agents, such asCD4 antagonists, CD2 antagonists and anti-IL-12 antibodies.

The immunomodulatory compound that may be co-administered with asynthetic CD40:CD154 binding interruptor compound to a subject with acondition associated with inappropriate CD154 activation may be anantibody that specifically binds to a protein selected from the groupconsisting of CD45, CD2, IL2R, CD4, CD8 and RANK Fc.

Formulation

In general, CD40:CD154 binding interrupter compounds of this inventionare suspended, dissolved or dispersed in a pharmaceutically acceptablecarrier or excipient. The resulting therapeutic composition does notadversely affect the recipient's homeostasis, particularly electrolytebalance. Thus, an exemplary carrier comprises normal physiologic saline(0.15 M NaCl, pH 7.0 to 7.4). Other acceptable carriers are well knownin the art and are described, for example, in Remington's PharmaceuticalSciences, Gennaro, ed., Mack Publishing Co., 1990. Acceptable carrierscan include biocompatible, inert or bioabsorbable salts, bufferingagents, oligo- or polysaccharides, polymers, viscoelastic compounds,such as hyaluronic acid, viscosity-improving agents, preservatives, andthe like.

A CD40:CD154 binding interrupter compound provided by this invention maybe administered in a pharmaceutically effective, prophylacticallyeffective or therapeutically effective amount, which is an amountsufficient to produce a detectable, preferably medically beneficialeffect on a subject at risk for or afflicted with a condition associatedwith inappropriate CD154 activation. Medically beneficial effectsinclude preventing, inhibiting, reversing or attenuating deteriorationof, or detectably improving, the subject's medical condition.

EXAMPLES

The following examples illustrate preferred embodiments of thisinvention related to novel CD40:CD154 binding interrupter compounds andthe use of these compounds to treat or prevent conditions associatedwith inappropriate CD154 activation in a subject. These examples shouldnot be construed as limiting: the examples are included for the purposesof illustration only.

The following method describes the preparation of a mass-codedcombinatorial library and the screening technology applied to obtainCD40:CD154 binding interrupter compounds disclosed in this invention.This method is advantageous because it can be used to identify chemicalcompounds that bind tightly to any biomolecule of interest, such asCD154. The screening technologies described can be miniaturized toprovide massive parallel screening capabilities.

Methods for producing mass-coded combinatorial libraries are disclosedin PCT patent application WO99/35109, published Jul. 15, 1999, thedisclosure of which is hereby incorporated by reference.

One method according to WO99/35109 allows identification of a member ormembers of a mass-coded combinatorial library which are ligands for abiomolecule, for example, a protein such as CD154. This method comprisesthe steps of: (1) contacting the biomolecule with the mass-codedmolecular library, whereby members of a mass-coded molecular librarywhich are ligands for the biomolecule bind to the biomolecule to formbiomolecule-ligand complexes and members of the mass-coded library whichare not ligands for the biomolecule remain unbound; (2) separating thebiomolecule-ligand complexes from the unbound members of the mass-codedmolecular library; (3) dissociating the biomolecule-ligand complexes;and (4) determining the molecular mass of each ligand to identify theset of n peripheral moieties present in each ligand.

The method of WO99/35109 allows rapid production of mass-codedcombinatorial libraries comprising large numbers of compounds. Themass-coding enables the identification of individual combinations ofscaffold and peripheral moieties by molecular mass. The resultinglibraries also allow the rapid identification of compounds which areligands for a given biomolecule, such as CD154.

One of the methods of WO99/35109 allows the preparation of a mass-codedset of compounds, such as a mass-coded combinatorial library. Thesecompounds are of the general formula X(Y)_(n), wherein X is a scaffold,each Y is a peripheral moiety and n is 4. The term “scaffold”, as usedin WO99/35109 and herein, refers to a molecular fragment to which fourperipheral moieties are attached via a covalent bond. The scaffold is amolecular fragment which is common to each member of the mass-coded setof compounds. The term “peripheral moiety”, as used in WO99/35109 andherein, refers to a molecular fragment which is bonded to a scaffold.Each member of the set of mass-coded compounds will include acombination of four peripheral moieties bonded to the scaffold and thisset of compounds forms a mass coded combinatorial library.

The term “mass” or “molecular mass”, as used in WO99/35109 and herein inrelation to combinatorial libraries, refers to the exact mass of amolecule or collection of chemical moieties in which each atom is themost abundant naturally occurring isotope for the particular element.Exact masses and their determination by mass spectrometry are discussedby Pretsch et al., Tables of Spectral Data for Structure Determinationof Organic Compounds, second edition, Springer-Verlag (1989), and Holdenet al., Pure Appl. Chem. 55 : 1119-1136 (1983), the contents of each ofwhich are incorporated herein by reference in their entirety.

A scaffold precursor represented by Formula IV, having four reactivegroups which are capable of reacting with a peripheral moiety precursorreactive group to form a covalent bond was chosen as a starting materialfor the synthesis of compounds according to this invention. Thatscaffold precursor, used in Scheme I (infra), is described in K. E.Pryor et. al. Tetrahedron 54: 4107-4124 (1998), the disclosure of whichis incorporated by reference herein. Scaffold precursors V-VI,respectively used in Schemes II and III (infra) were synthesizedaccording to the reaction scheme shown by Pryor et. al., followed byadditional modification with conventional chemical protectingtechniques.

A peripheral moiety precursor is a compound which includes a reactivegroup which is complementary to the reactive groups of the scaffoldprecursor. In addition to the reactive group, a peripheral moietyprecursor can include a wide variety of structural features. Forexample, the peripheral moiety precursor can include one or morefunctional groups in addition to the reactive group. Any additionalfunctional group should be appropriately masked or not interfere withthe reaction between the scaffold precursor and the peripheral moietyprecursor. Suitable protecting groups for masking are known in the artfor a variety of functional groups (Greene and Wuts, Protective Groupsin Organic Synthesis, second edition, New York: John Wiley and Sons(1991), and third edition, New York: John Wiley and Sons (1999), thedisclosures of both of which are incorporated herein by reference).Particularly useful protecting groups include t-butyl esters and ethers,acetals, trityl ethers and amines, acetyl esters, trimethylsilyl ethersand trichloroethyl ethers and esters. In addition, two peripheral moietyprecursors should not react together under the conditions employed. Forexample, a subset of peripheral moiety precursors can include, inaddition to the reactive groups, functionalities selected from groupsspanning a range of charge, hydrophobicity/hydrophilicity, and sizes.For example, the peripheral moiety precursor can include a negativecharge, a positive charge, a hydrophilic group or a hydrophobic group.

For the present purposes, two reactive groups are complementary if theyare capable of reacting together to form a covalent bond. In a preferredembodiment, the bond forming reactions occur rapidly under ambientconditions without substantial formation of side products. Preferably, agiven reactive group will react with a given complementary reactivegroup exactly once.

Complementary electrophilic and nucleophilic groups include any twogroups which react via nucleophilic substitution under suitableconditions to form a covalent bond. An example of a suitableelectrophilic group is an acid chloride. A suitable nucleophilic groupinclude primary and secondary amino groups.

Illustrated below in Table II are examples of suitable peripheral moietyprecursors. Each peripheral moiety precursor includes a primary orsecondary amino group which reacts with the scaffold precursor to forman amide bond. TABLE II BB BB BB ID* MOLSTRUCTURE Mass ID* MOLSTRUCTUREMass ID* MOLSTRUCTURE Mass  11

175.136  61

107.073 138

155.05  13

113.12 115

147.053 140

168.163  15

127.136 116

75.032 141

144.163  16

127.136 117

131.095 157

116.131  17

85.0891 118

131.095 161

179.095  27

114.116 119

146.106 201

119.058  36

75.0684 120

165.079 204

136.105  45

87.0684 122

115.063 214

141.035  50

220.121 124

105.043 226 —NH₂ 31.0422  52

154.147 127

117.079 243

87.1048 245

102.116 300

135.105 387

147.105 256

151.063 301

137.084 388

147.105 270

89.0477 307

165.079 396

151.1 277

184.984 315

200.062 419

103.063 283

179.095 318

145.11 424

157.11 286

133.089 323

151.063 465

111.08 287

133.089 350

116.131 466

160.1 288

137.084 353

130.147 475

101.084 290

165.115 370

127.046 500

185.061 292

135.105 385

101.048 514

142.111 533

114.116 587

103.063 597

130.111 551

114.079 588

182.178 598

266.178 564 NH₃ 17.0266 589

142.147 599

116.095 580

146.069 590

174.112 600

183.105 581

132.054 591

181.074 601

213.015 582

155.07 592

149.051 602

131.095 583

211.136 593

156.159 603

171.126 584

133.089 594

157.089 604

117.079 585

149.12 595

149.12 605

184.121 586

185.142 596

164.095 606

170.142 607

182.142 617

213.015 634

210.064 608

168.163 618

213.015 635

157.11 609

168.126 619

203.027 636

209.105 610

128.131 625

131.095 638

157.11 611

186.083 628

133.089 639

131.095 612

172.103 629

133.089 613

210.173 630

129.079 614

196.194 631

143.095 615

154.111 632

155.095 616

140.131 633

177.079*Identification number of the peripheral moiety precursors.

The compounds within the combinatorial library set are mass-coded as aresult of the selection of a subset of suitable peripheral moietyprecursors. The mass-coded set of compounds is synthesized in solutionas a combinatorial library.

In one embodiment, the scaffold precursor is contacted with all membersof the peripheral moiety precursor subset simultaneously. In general, ascaffold precursor having four reactive groups will be contacted with atleast about 4 molar equivalents relative to the scaffold precursor ofperipheral moiety precursors from the selected subset. For example, thescaffold precursor can be contacted with a solution comprising eachmember of the subset in approximately equal concentrations. In oneembodiment, the reaction protocol shown below (Scheme I) was used.

In another embodiment, the reaction protocol shown below (Scheme II) wasused.

In another embodiment, the reaction protocol shown below (Scheme III)was used.

Following the reaction of each scaffold precursor reactive group with aperipheral moiety precursor, any peripheral moiety having a protected,additional functional group can be deprotected using methods known inthe art.

The ability to identify individual scaffold plus peripheral moietycombinations derived from such a mixture is a consequence of themass-coding of the library and the ability of mass spectrometry toidentify a molecular mass. This allows the identification of individualscaffold plus peripheral moiety combinations within the set which have aparticular activity, such as binding to a particular biomolecule, suchas CD154.

In one embodiment, a method is provided for identifying a compound orcompounds within a mass-coded combinatorial library which bind to, orare ligands for, a biomolecule, such as a CD154. The mass-codedcombinatorial library can be produced, for example, by the methoddisclosed above. The target biomolecule, such as CD154, is contactedwith the mass-coded combinatorial library, and, if any members of thelibrary are ligands for a target biomolecule, biomolecule-ligandcomplexes form. Compounds which do not bind the target biomolecule areseparated from the biomolecule-ligand complexes. The biomolecule-ligandcomplexes are dissociated and the ligands are separated and theirmolecular masses are determined by mass spectrometry. Due to the masscoding of the combinatorial library, a given molecular mass ischaracteristic of a unique combination of peripheral moieties or only asmall number of such combinations. Thus, a ligand's molecular massallows the determination of its composition.

Once single ligands are identified by the above-described process,various levels of analysis can be applied to yield SAR information andto guide further optimization of the affinity, specificity andbioactivity of the ligand. For ligands derived from the same scaffold,three-dimensional molecular modeling can be employed to identifysignificant structural features common to the ligands, therebygenerating families of small-molecule ligands that presumably bind at acommon site on the target biomolecule.

To identify a consensus, highest affinity ligand for a particularbinding site, this analysis should include a ranking of the members of agiven ligand family with respect to their affinities for the target.This process can provide this information by identifying both low andhigh affinity ligands for a target biomolecule in one experiment.

Given that each mass-coded set of compounds is synthesized with alimited number of peripheral moiety precursors, the described approachcan, in certain cases, identify a superior ligand which combinesstructural features of molecules synthesized in separate libraries.

When possible, the analysis of ligand structural features is based oninformation regarding the target biomolecule's structure, wherein thehypothetical consensus ligand is computationally docked with theputative binding site. Further computational analysis can involve adynamic search of multiple lowest energy conformations, which allowscomparison of high affinity ligands that are derived from differentscaffolds. The end goal is the identification of both the optimalfunctionality and the optimal vectorial presentation of the peripheralmoieties that yields the highest binding affinity/specificity. This mayprovide the basis for the synthesis of an improved, second-generationscaffold.

An advantage of the method discussed above is that it can be used toidentify chemical compounds that bind tightly to any biomolecule ofinterest, such as CD154.

Screening a Mass-Coded Combinatorial Library for Novel CD40:CD154Binding Interruptors

Methods for identifying compounds in a chemical mixture are described inthe PCT patent application WO00/22649, published Apr. 20, 2000, thedisclosure of which is hereby incorporated by reference. These methodsand the methods disclosed in WO99/35109 were used to identify compoundsthat bind CD154 from a mixture of compounds (a mass-coded combinatoriallibrary).

The WO00/22649 application relates generally to Mass Spectrographicanalysis, and more specifically to the identification of organiccompounds in complex mixtures of organic compounds.

The mass coded combinatorial library was prepared by-treating thereactive scaffold precursors (shown in Formulae IV-VI) with pools ofbuilding blocks shown in Table II using the reaction protocols shown inSchemes I-III.

The mass-coded combinatorial library was screened for novel CD40:CD154binding interrupters according the the methods discussed herein. Thelibrary was first screened for compounds that bind CD154. Thosecompounds that interact with CD154 were identified, isolated and theirchemical compositions were verified with mass spectrometry. Thecompounds that interact with CD154 can then each be synthesized on alarger scale following the method described below.

The compounds were then screened for the ability to inhibit CD40:CD154interaction. A person skilled in the art is well aware of conventionalassays to assess whether the compounds designed according to thisinvention bind specifically to CD154 and whether the compounds of thisinvention interrupt CD40:CD154 interaction. These assays, for example,detect the extent to which B cells are activated by activated T cellsvia the interaction between CD154 and CD40. For example, monitoring ofCD23 levels on B cells, or secretion of immunoglobulins by B cells isindicative of activation of B cells by activated T cells via theinteraction between CD40 and CD154. See, e.g., U.S. Pat. No. 5,474,771.Accordingly, examples of such assays are: the assay of Example 7, an invitro assay for T cell activation of B cells; an in vitro assay forimmunoglobulin production by B cells and an in vivo assay for inhibitionof a humoral immune response.

Once a compound is discovered by the methods described above,substitutions may then be made by conventional methods in some of itsatoms or side groups to further improve or modify its binding and/orinhibition properties. Such substituted chemical compounds may then beanalyzed for binding to CD154 and inhibition of CD40:CD154 interaction.

Example 1 Chemical Synthesis of Compound 7

Step 1: A mixture of chelidamic acid monohydrate (1.00 g, 4.97 mmol) andphosphorus pentabromide (8.45 g, 19.6 mmol) was warmed to 90° C. for 3hours (h). Chloroform (100 mL) was added to the warm reaction mixtureand the resulting slurry was filtered. The filtrate was concentrated invacuo to give a pink solid. The solid was dissolved in ethanol (vigorousreaction), stirred for 0.5 h and then concentrated in vacuo to give ayellow solid. The solid was dissolved in ether (250 mL), washed withsaturated aqueous sodium bicarbonate (50 mL×2), dried (MgSO₄) andconcentrated in vacuo to give 1.30 g of a white solid.

Step 2: A solution of KOH (0.83 g, 3.31 mmol) in anhydrous ethanol (40mL) was added dropwise to a solution of the compound from step 1 (3.85g, 12.7 mmol) in anhydrous ethanol (100 mL) over the course of 3 h. Awhite precipitate formed. The unstirred slurry was allowed to sit atroom temperature (RT) for 7 h and then at 0° C. overnight. The slurrywas filtered and the resulting white solid dissolved in water (100 mL),cooled to 0° C. and acidified to pH 1 with 1N HCl. The slurry wasfiltered and the resulting white solid dried in vacuo to give 3.00 gwhite solid.

Step 3: Catalytic DMF and 2.0 M oxalyl chloride/dichloromethane (4.5 mL,9.0 mmol) were added to a solution of the compound from step 2 (1.20 g,4.40 mmol) in dichloromethane at RT. The reaction solution was stirreduntil bubbling ceased and was then concentrated in vacuo. The residuewas dissolved in toluene (20 mL) and added dropwise to a solution ofL-cyclohexylglycine tbutyl ester.HCl (1.20 g, 4.80 mmol) in pyridine (10mL) at RT. The reaction was stirred overnight at RT, diluted with ether,washed with water, dried (MgSO₄) and concentrated in vacuo to give anoil. The oil was purified via flash column chromatography (95:5methylene chloride/ethyl acetate) to give 1.01 g of a colorless oil.

Step 4: A solution of the compound from step 3 (0.500 g, 1.07 mmol),bis(pinacolate)diboran (0.300 g, 1.18 mmol),[1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)•methylenechloride (0.026 g, 0.032 mmol) and potassium acetate (0.315 g, 3.21mmol) in anhydrous DMF (7 mL) was warmed to 80° C. under a nitrogenatmosphere for 2 h. The reaction was then cooled to RT, diluted withethyl acetate (70 mL), washed with 5% citric acid (25 mL), 50 sodiumbicarbonate (25 mL) and water (25 mL), dried (MgSO₄) and concentrated invacuo to give a brown oil.

Step 5: 3-Phenyl-1-propylamine (4.0 mL, 28.1 mmol) was added dropwise toconcentrated nitric acid at 0° C. The reaction was allowed to warm to RTovernight. The reaction was warmed to 50° C. for 2 h, concentratednitric acid (10 mL) was added and the reaction was warmed for another 2h at 50° C. The reaction was cooled to 0° C. and poured into ice water.The yellow precipitate was isolated via filtration, washed with waterand air dried to give 1.30 g of a yellow solid. ¹H NMR indicatedformation of the para isomer only.

Step 6: Catalytic DMF and 2.0 M oxalyl chloride/dichloromethane (4.5 mL,9.0 mmol) were added to a solution of the compound from step 2 (1.20 g,4.40 mmol) in dichloromethane at RT. The reaction solution was stirreduntil bubbling ceased and concentrated in vacuo. The residue wasdissolved in toluene (20 mL) and added dropwise to a solution of thecompound from step 5 (1.20 g, 4.93 mmol) in pyridine (10 mL) at RT. Thereaction was stirred overnight at RT, diluted with ether, washed withwater, dried (MgSO₄) and concentrated in vacuo to give an oil. The oilwas purified via flash column chromatography (90:10 methylenechloride/ethyl acetate) to give 0.51 g of a yellow solid.

Step 7: 2 M aqueous sodium carbonate (2.70 mL, 5.4 mmol) was added to asolution of the compound from step 4, the compound from step 6 (0.51 g,1.17 mmol) and[1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)•methylenechloride (0.026 g, 0.032 mmol) in anhydrous DMF. The reaction was warmedto 80° C. overnight. It was then cooled to RT, diluted with ethylacetate, washed with water and brine, dried (MgSO₄) and concentrated invacuo to give a brown oil.

Step 8: Lithium hydroxide was added to a solution of the compound fromstep 7 in 2:1 THF/water at RT. When all starting ester was consumed viaHPLC analysis, the reaction was diluted with cold water and washed withethyl acetate. The aqueous phase was saturated with ammonium chlorideand extracted with THF. The combined organic phases were dried (MgSO₄)and concentrated in vacuo. The resulting solid was washed with water andair dried to give 1.20 g of a dark solid. (approx. 50% desired material)

Step 9: S-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine (0.80 g, 5.2 mmol),HATU (2.0 g, 5.3 mmol) and diisopropylethyl amine (1.8 mL, 10.3 mmol)were added to a solution of the compound from step 8 (1.2 g) inanhydrous DMF (25 mL) at RT. The reaction was stirred at RT overnight.The volatiles were removed in vacuo, the residue dissolved in ethylacetate and washed with water. The organic phase was dried (MgSO₄),concentrated in vacuo and purified via flash column chromatography(100:10:2 ethyl acetate/methanol/triethyl amine) to give 0.650 g of abrown solid.

Step 10: A solution of the compound from step 9 (0.030 g) in 2:1TFA/methylene chloride (6 mL) was stirred at RT for 3 h. The reactionwas then concentrated in vacuo and purified via reverse phase HPLC(acetonitrile/water) to give a white solid.

Example 2 Chemical Synthesis of Compound 8

Step 1: steps 1-9 of EXAMPLE 1 were carried out.

Step 2: Catalytic 10% Pd/C was added to a solution of the compound fromstep 1 (0.100 g) in ethyl acetate. The reaction was stirred under ahydrogen atmosphere (1 atm) overnight. The reaction was then centrifugedand the supernatant decanted and concentrated in vacuo.

Step 3: A solution of the compound from step 2 (0.020 g) in 2:1TFA/methylene chloride (6 mL) was stirred at RT for 2 h. The reactionwas then concentrated in vacuo and purified via reverse phase HPLC(acetonitrile/water) to give an off-white solid.

Example 3 Chemical Synthesis of Compound 4

Step 1: steps 1 and 2 of EXAMPLE 1 were carried out.

Step 2: Catalytic DMF and 2.0 M oxalyl chloride/dichloromethane (3.0 mL,6.0 mmol) were added to a solution of the compound from step 1 (2.72 g,2.64 mmol) in dichloromethane (20 mL) at RT. The reaction solution wasstirred until bubbling ceased, diluted with toluene (40 mL) and reducedin volume in vacuo. A solution of 3-phenyl-1-propylamine (1.7 mL, 11.9mmol) in pyridine (8 mL) was added and the resulting solution wasstirred at RT overnight. The reaction solution was diluted with ethylacetate (300 mL), washed with water (150 mL), 1N HCl (150 mL×3) andwater (150 mL), dried (MgSO₄) and concentrated in vacuo to give an oil.The oil was dissolved in 1:1 saturated aqueous lithium hydroxide/THF andstirred overnight at RT. The reaction was then acidified with 1N HCl,extracted with ethyl acetate, dried (MgSO₄) and concentrated in vacuo togive 3.456 g of an oil.

Step 3: A solution of the compound from step 2 (3.456 g, 9.515 mmol),bis(pinacolate)diboran (2.658 g, 10.54 mmol),[1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)•methylenechloride (1.029 g, 1.26 mmol) and potassium acetate (3.88 g, 39.5 mmol)in anhydrous DMF (60 mL) was warmed to 80° C. under a nitrogenatmosphere for 2.5 h. The reaction was then cooled to RT, diluted withethyl acetate (500 mL) and extracted with 1N sodium hydroxide (200 mL).The aqueous phase was washed with ethyl acetate (250 mL×2), acidifiedwith concentrated HCl to pH 0 and extracted with ethyl acetate (500mL×2). The combined organic phases were dried (MgSO₄) and concentratedin vacuo to give an oil which was slurried with ether. The resultantlight brown solid was filtered and air dried to give 1.48 g of product.The mother liquor was concentrated in vacuo to give 1.78 g of product.

Step 4: L-cyclohexylglycine tbutyl ester.HCl (1.717 g, 6.87 mmol), HATU(2.618 g, 6.89 mmol) and diisopropylethyl amine (4.00 mL, 23.0 mmol)were added to a solution of the compound from Example 1, step 2 (1.704g, 6.24 mmol) in anhydrous DMF (10 mL) at RT. The reaction was stirredat RT for 2.5 h. The reaction was diluted with saturated aqueous sodiumbicarbonate (50 mL) and extracted with ether (150+100 mL). The organicphases were washed with water (100+50 mL), dried (MgSO₄) andconcentrated in vacuo to give 2.994 g of a yellow oil.

Step 5: Saturated aqueous lithium hydroxide (3 mL) was added to asolution of the compound from step 4 (2.994 g, 6.24 mmol) in THF (10 mL)at RT. After 1 h, saturated aqueous sodium bicarbonate (50 mL) wasadded. The slurry was then cooled to 0° C., acidified with 1N HCl andextracted with ethyl acetate (400+100 mL). The combined organic phaseswere dried (MgSO₄) to give 2.238 g of a light yellow powder.

Step 6: 2 M aqueous sodium carbonate (7.55 mL, 15.1 mmol) was added to asolution of the compound from step 3 (0.820 g, 2.00 mmol), the compoundfrom 5 (0.883 g, 2.00 mmol) and[1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)•methylenechloride (0.267 g, 0.327 mmol) in anhydrous DMF (10 mL). The reactionwas warmed to 85° C. for 2.5 h. It was then cooled to RT, diluted withethyl acetate (400 mL), and extracted with water (150 mL). The aqueousphase was washed with ethyl acetate (100 mL) and acidified with 1N HCl.The resulting slurry was extracted with ethyl acetate (400+100 mL), thecombined organic phases were dried (MgSO₄) and concentrated in vacuo togive a white solid. The white solid was slurried with ether, filteredand air dried to give 0.932 g of product. Concentration in vacuo of themother liquor produced another 0.359 g of desired product.

Step 7: S-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine (0.200 mL, 1.23 mmol),HATU (0.434 g, 1.14 mmol) and diisopropylethyl amine (0.300 mL, 1.72mmol) were added to a solution of the compound from step 6 (0.359 g,0.557 mmol) in anhydrous DMF (3 mL) at RT. The reaction was stirred atRT for 3 h. The reaction was diluted with ethyl acetate (300 mL), washedwith saturated aqueous sodium carbonate (50 mL×2), dried (MgSO₄) andconcentrated in vacuo to give 0.675 g of an oil.

Step 8: TFA (4 mL) and water (0.100 mL) was added to a solution of thecompound from step 7 (0.675 g) in acetonitrile (3.0 mL). After 4 h, thereaction was concentrated in vacuo and purified via reverse phase HPLC(acetonitrile/water) to give 0.123 g of product.

Example 4 Chemical Synthesis of Compound 10

Step 1: steps 1 and 2 of EXAMPLE 1 were carried out.

Step 2: S-(+)-1-(2-pyrrolidinylmethyl)pyrrolidine (5.3 g, 34.4 mmol),HATU (17.5 g, 46.0 mmol) and diisopropylethyl amine (17 mL, 97.6 mmol)were added to a solution of the compound from step 1 (8.50 g, 31.1 mmol)in anhydrous DMF (100 mL) at RT. The reaction was stirred at RT for 0.75h. The reaction was diluted with ethyl acetate, washed with saturatedaqueous sodium bicarbonate and water, dried (MgSO₄) and concentrated invacuo to give 13 g of the desired product.

Step 3: The compound from step 2 (6.5 g, 15.8 mmol) was dissolved in 1:1THF/saturated aqueous lithium hydroxide (50 mL) at RT. After 2 h, thereaction was concentrated in vacuo, acidified with saturated ammoniumchloride and concentrated in vacuo. The resulting solid was slurried inTHF and filtered. The filtrate was concentrated in vacuo to give thedesired product.

Step 4: L-cyclohexylglycine tbutyl ester.HCl (3.75 g, 15.0 mmol), HATU(7.5 g, 19.7 mmol) and diisopropylethyl amine (10 mL, 57 mmol) wereadded to a solution of the compound from step 3 in anhydrous DMF (200mL) at RT. The reaction was stirred at RT for 0.5 h. The reaction wasdiluted with ethyl acetate, washed with saturated sodium bicarbonate andwater, dried (MgSO₄) and concentrated in vacuo to give the desiredproduct.

Step 5: A solution of the compound from step 2 (6.5 g, 15.8 mmol),bis(pinacolate)diboran (4.4 g, 17.3 mmol),[1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)•methylenechloride (0.388 g, 0.475 mmol) and potassium acetate (4.7 g, 47.9 mmol)in anhydrous DMF (100 mL) was warmed to 80° C. under a nitrogenatmosphere for 2 h. The reaction was then cooled to RT, diluted withethyl acetate, washed with 5% citric acid and 5% sodium bicarbonate,dried (MgSO₄) and concentrated in vacuo to give a brown oil.

Step 6: 2 M aqueous sodium carbonate (40 mL, 80 mmol) was added to asolution of the compound from step 4, the compound from step 5 and[1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)•methylenechloride in anhydrous DMF (100 mL). The reaction was warmed to 80° C.overnight. It was then cooled to RT, acidified with saturated ammoniumchloride and extracted with THF (10×). The combined organic phases weredried (MgSO₄) and concentrated in vacuo to give the carboxylic acid. Onehalf of the material was used as crude material and half was purifiedvia reverse phase HPLC (acetonitrile/water).

Step 7: Diethylcyanomethylphosphonate (2.7 mL, 16.7 mmol) was addeddropwise to a slurry of NaH/mineral oil (0.730 g, 19.0 mmol) inanhydrous THF (10 mL) at RT. After the initial bubbling ceased, thereaction was warmed to reflux for 0.25 h. A solution of3-nitrobenzophenone (1.90 g, 8.36 mmol) in anhydrous THF (20 mL) wasadded dropwise and the reaction warmed at reflux for 3 h. After coolingto RT, the reaction was poured into citric acid solution and extractedwith ethyl acetate (2×). The combined organic phases were washed withwater, dried (MgSO₄) and concentrated in vacuo. The residue was purifiedvia flash column chromatography (methylene chloride) to give 1.81 g ofan oil.

Step 8: A slurry of 10% Pd/C (0.500 g) and the compound from step 7(1.81 g) in ethyl acetate (50 mL) was subjected to a hydrogen atmosphere(60 psi) at RT. After 18 h the reaction slurry was filtered andconcentrated in vacuo to give 1.50 g of the desired product.

Step 9: 1M Borane tetrahydrofuran complex/THF (28 mL, 28 mmol) was addedto a solution of the compound from step 8 (1.50 g) in anhydrous THF. Thereaction was warmed to reflux for 3 days, cooled to RT and quenched withmethanol. After the initial bubbling had ceased, more methanol (15 mL)was added and the reaction warmed to reflux for 0.5 h. The reaction wasconcentrated in vacuo, diluted with methanol, filtered and the filtrateconcentrated in vacuo to give the desired product.

Step 10: The compound from step 9 (0.127 g, 0.561 mmol), HATU (0.110 g,0.289 mmol) and diisopropylethyl amine (excess) were added to a solutionof the compound from step 6 in anhydrous DMF (5 mL) at RT. The reactionwas stirred at RT for 0.5 h. The reaction was diluted with ethylacetate, washed with saturated sodium bicarbonate and water, dried(MgSO₄), concentrated in vacuo and purified via reverse phase HPLC togive 0.005 of the desired product.

Step 11: A solution of the compound from step 10 in 1:1 TFA/methylenechloride was stirred at RT for 2 h, concentrated in vacuo and purifiedvia reverse phase HPLC to give 0.0015 g of the desired product.

Example 5 Chemical Synthesis of Compound 11

Step 1: 4-nitrodiphenylmethane (1.00 g, 4.69 mmol) was slowly added to aslurry of potassium t-butoxide (1.05 g, 9.36 mmol) in anhydrous DMF (20mL) at RT. After 5 min. t-butyl bromoacetate (1.0 mL, 6.77 mmol) wasadded and the reaction stirred overnight at RT. The reaction was dilutedwith ether, washed with 5% citric acid solution and water, dried (MgSO₄)and concentrated in vacuo to give an oil. The oil was purified via flashcolumn chromatography (60:40 methylene chloride/hexanes) to give acolorless solid.

Step 2: The compound from step 1 was dissolved in 1:1 TFA/methylenechloride (20 mL) at RT. After 0.5 h the reaction solution wasconcentrated in vacuo. The residue was dissolved in anhydrous methylenechloride, DMF (1 drop) and 2M oxalyl chloride/methylene chloride (10 mL,20 mmol) was added. After 0.5 h bubbling ceased, and the reaction wasconcentrated in vacuo. The residue was dissolved in toluene (25 mL) andammonia gas was bubbled into the reaction for 0.5 h. The reaction wasdiluted with ethyl acetate, washed with sodium bicarbonate solution,dried (MgSO₄) and concentrated in vacuo to give 0.560 g of a colorlessoil.

Step 3: 1M Borane tetrahydrofuran complex/THF (10 mL, 10 mmol) was addedto a solution of the compound from step 2 (0.560 g) in anhydrous THF (10mL). The reaction was warmed to reflux overnight, cooled to RT andquenched with methanol. After the initial bubbling had ceased, moremethanol (10 mL) was added and the reaction warmed to reflux for 0.5 h.The reaction was concentrated in vacuo, and purified via flash columnchromatography (80:20:2 ethyl acetate/methanol/triethylamine) to give0.130 g of a light yellow oil.

Step 4: Diisopropylethylamine (0.200 mL, 1.12 mmol) was added to asolution of the compound from step 3 (0.130 g, 0.507 mmol), the compoundfrom Example 4, step 6 (0.250 g, 0.313 mml) and HATU (0.200, 0.526 mmol)in anhydrous DMF (10 mL) at RT., After 0.5 h the reaction wasconcentrated in vacuo, dissolved in ethyl acetate, washed with saturatedsodium bicarbonate and water, dried (MgSO₄), concentrated in vacuo andpurified via reverse phase HPLC (acetonitrile/water) to give 0.140 g ofan off-white powder.

Step 5: A solution of the compound from step 4 (0.010 g) in 1:1TFA/methylene chloride (4 mL) was stirred at RT for 2 h. The reactionwas then concentrated in vacuo and purified via reverse phase HPLC(acetonitrile/water) to give a white solid.

Example 6 Chemical Synthesis of Compound 9

Step 1: 2M (trimethylsilyl)diazomethane/hexanes (0.100 mL, 0.200 mmol)was added to a solution of the compound from Example 3, step 8 (0.020 g,0.023 mmol) in acetonitrile (0.5 mL). The reaction was stirred at RT for0.25 h, concentrated in vacuo and purified via reverse phase HPLC(acetonitrile/water) to give a white solid.

Example 7 Binding Assay to Assess whether a compound Binds CD154 andInhibits CD40:CD154 Interaction

1) Ninety-six well plates of Nunc Maxisorp were coated with 100 μl of5-10 μg/ml human CD40-Ig produced in Pichia cells, (in PBS pH 7.2) perwell, covered with plate sealers (Costar, Cat. No. 3095) and storedovernight at 4° C.

2) The CD40-Ig-coated plates were shook out and blotted dry.

3) The CD40-Ig-coated plates were blocked with 300 μl per well of PBS(0.05% Tween-20, 1% BSA, pH 7.2) at RT for one hour or in therefrigerator for up to 3 weeks.

4) The CD40-Ig-coated plates were washed 3× with PBS+0.05% Tween-20 pH7.4 with plate washer at RT.

5) The plates were optionally blotted dry.

6) Primary Screening of compounds:

-   -   a. Compounds were titrated in 100 mM Hepes+0.005% BSA pH 7.2 in        Titertube (Bio-Rad Laboratories) and 50 μl of titrated compounds        was then added into each well of the plate. IC50 parameters:        -   One 7 point curve per compound was generated where each            point was run in triplicate. A 2× dilution series was used            to generate the curve.        -   Top concentration in assay of test compound was 125 μM.    -   b. Fifty μl/well of 0.07-1.6 μg/ml biotin-CD154 in 100 mM        Hepes+0.005% BSA (pH 7.2) was added per well. Final        concentration of biotin-CD154 was 0.035-0.08 μg/ml. It was mixed        with compound in each well in the plate.    -   c. The plates were covered with plate sealer and incubated for        one-hour at RT.

7) The plates were washed 3× with plate washer with PBS+0.05% Tween-20(pH 7.4) at RT.

8) The plates were optionally blotted dry.

9) 100 μl of a 1/300,000 dilution of HRP-Strep/Avidin (Jackson ImmunoResearch Code3 #016-030-084) was added to each well. Dilution bufferwas: PBS+0.05% Tween-20. The plates were covered with plate sealer andincubated for one hour at room temperature.

10) The plates were washed with plate washer 3× at RT.

11) The plates were optionally blotted dry.

12) 100 μl/well of TMB(1-component) (Kirkegarrd & Perry Labs, Prod#50-76-04) was added to each well and incubated at RT about 10 min.

13) 100 μl/well of 0.18M H₂SO₄ was added to stop reaction in the plates.

14) The plates were read within 30 minutes of stopping the reaction at450 nM on a microplate reader (Molecular Devices Co.).

Compounds that bound to CD154 and blocked CD40:CD154 interactionprevented CD154 from binding to immobilized CD40-Ig and gave a lowabsorbance reading.

Controls:

-   1. Total reaction: Reaction well contained 50 μl of 100 mM    Hepes/0.005% BSA (no test compound)-   2. Positive control: Reaction well contained 10 μl of 1 mg/ml 5c8    (anti-CD154 mAb) and 50 μl of 100 mM Hepes/0.005% BSA.-   3. Blank control: Reaction well contained 100 μl 100 mM Hepes/0.005%    BSA.-   4. Compound control: Titration series in triplicate of    BIO-002108-00.

BIO-002108-00 is one of the derivatives of suramin (CalBiochem catalognumber 574625). It has been demonstrated that BIO-002108-00, as well asother derivatives of suramin, is a CD40:CD154 binding interrupter.BIO-002108-00 has an IC50 in the A range (less than 50 μM) forCD40:CD154 binding interruption as determined by this assay.BIO-002108-00 has an IC50 of greater than 125 μM (average value) forTNF-α:TNFRp60 binding interruption. BIO-002108-00 has the followingstructure:

It was synthesized by the following method:

Step 1

2M (Trimethylsilyl)diazomethane/hexanes (50 mL, 100 mmol) was addeddropwise to a solution of tech.

3-amino-2-naphthoic acid (3 g, 12.8 mmol) in methanol (300 mL) at RT. Agrey precipitate was formed. The reaction was centrifuged, thesupernatant was decanted, concentrated in vacuo and purified via flashcolumn chromatography (methylene chloride) to give 1.78 g of a yellowcrystalline solid.

Step 2

A solution of the compound from step 1 (0.205 g, 1.02 mmol) and3-nitrobenzenesulfonyl chloride (0.250 g, 1.13 mmol) in pyridine (5 mL)was stirred at RT for 0.5 h. The reaction was then poured into 1Naqueous hydrochloric acid (100 mL) and extracted with methylene chloride(2×100 mL). The combined organic phases were washed with 1N aqueoushydrochloric acid (100 mL) and water (100 mL), dried (MgSO₄) andconcentrated in vacuo to give 0.380 g of a reddish syrup.

Step 3

10% Pd/C (0.500 g) was added to a solution of the compound from step 2(0.380 g) in 1:1 ethyl acetate/methanol (50 mL). The reaction wasstirred under a hydrogen atmosphere (1 atm) for 16 h. The reaction wascentrifuged, the supernatant decanted, concentrated in vacuo andpurified via flash column chromatography (95:5 methylene chloride/ethylacetate) to give 0.278 g of a white solid.

Step 4

A solution of the compound from step 3 (0.060 g, 0.169 mmol) and1,1′-carbonyldiimidazole (0.030 g, 0.185 mmol) in anhydrous THF (1 mL)was refluxed overnight. The reaction was then cooled to RT, concentratedin vacuo and purified via reverse phase HPLC (acetonitrile/water) togive 0.018 g of a white solid.

Step 5

Lithium hydroxide (0.100 g, 2.28 mmol) was added to a solution of thecompound from step 4 (0.018 g) in 2:1:1 THF/methanol/water (20 mL).After stirring overnight at RT, the reaction was poured into 1N aqueoushydrochloric acid (25 mL) and extracted with ethyl acetate (2×50 mL).The combined organic phases were dried (MgSO₄) and purified via reversephase HPLC (acetonitrile/water) to give 0.001 g white solid. MS (ESP-)709.09 (M-1)

Example 8 Binding Assay to Assess whether a Compound Binds to TNF-α andInhibits TNF-α/TNFRp60 Interaction

1) Ninety-six well plates of Nunc Maxisorp were coated with 100 μl/wellof 0.6 ug/ml tumor necrosis factor receptor p60 (TNFRp60) (Browning, J.L.; Douglas, I.; Ngam-ek, A.; Bourdon, P. R.; Ehrenfels, B. N.;Miatkowski, K.; Zafari, M.; Yampaglia, A. M.; Lawton, P.; Meier, W.;Benjamin, C. P.; Hession, C. J. Immunol. 1995, 154, 33-46) in PBS, pH7.2, covered with plate sealers (Costar, Cat. No. 3095) and storedovernight at 4° C.

2) The coated plates were shook out and blotted dry.

3) TNFRp60-coated plates were blocked with 300 μl per well of PBS (0.05%Tween-20, 1% BSA, made from media prep) at 4° C. for one hour. Plateswere stored for 3 weeks at 4° C.

4) The plates were washed 3× with PBS+0.05% Tween-20) by plate washer atroom temperature.

5) The plates were optionally blotted dry.

6) Primary Screening of compounds

Compounds were titrated in 100 mM Hepes+0.005% BSA, pH 7.2 in Titertube(Bio-Rad Laboratories) and then 50 μl of titrated compounds was addedinto each well of the plate.

IC50 parameters: One 7 point curve per compound was generated where eachpoint was run in triplicate. A 2× dilution series was used to generatethe curve. The top concentration in assay of test compound was 125 μM.

50 μl/well of 8 ng/ml Biotin-TNF-α (R&D System) in 100 mM Hepes+0.005%BSA, pH 7.2 were added. The final concentration of Biotin-TNFα was 4ng/ml. It was mixed with compound in each well in the plate. The plateswere covered with sealer and incubated one hour at RT.

7) The plates were washed 3× by plate washer with PBS+0.05% Tween-20 atRT.

8) The plates were optionally blotted dry.

9) 100 μl of Streptavidin-HRP (diluted at 1:300,000 in PBS+0.05%Tween-20) was added to each well. The plates were covered with sealerand incubated one hour at RT.

10) The plates were washed by plate washer 3× at RT.

11) The plates were optionally blotted dry.

12) 100 μl/well of TMB(1-component) (Kirkegarrd & Perry Labs, Prod#50-76-04) were added at RT and incubated about 10 min in darkenvironment.

13) 100 μl/well of 0.18M H₂SO₄ was added to stop the reaction.

14) The plates were read within 30 minutes of adding stop reagent at 450nM by microplate reader (Molecular Devices Co.)

Controls:

-   -   Total reaction: Reaction well contained 50 μl of 100 mM        Hepes/0.005% BSA (no test compound).    -   Positive control: Reaction well contained 10 μl of 1.5 mg/ml        anti-TNF-α and 50 ul of 100 mM Hepes/0.005% BSA.    -   Blank control: Reaction well contains 100 μl 100 mM Hepes/0.005%        BSA.    -   Compound control: Titration series in triplicate of        BIO-002108-00.

Compounds that blocked TNF-α/TNFRp60 interaction prevented TNF-α frombinding to immobilized TNFRp60 and gave a low absorbance reading.

The above-described assay may also be carried out using TNFRp60-Fc inplace of TNFRp60.

In addition to the foregoing assays (Examples 7-8), the followingbinding assay may be used to assess activity of compound according tothis invention.

Example 9

ICAM Assay

1. Set up wells containing:

-   -   Different concentrations of compounds in 50 uL of RPMI (10%        serum, 4 mM glutamine, pen/strep)    -   0.5 ug of CD40 ligand in 50 uL    -   2×10⁵ 2G6 cells in 100 uL

Set up groups of cells only

-   -   Cells+compound    -   Cells+ligand    -   Cells+ligand+10 ug of 5c8 mAb

Each group should have a final volume in the well of 200 uL. If theconcentration of DMSO>0.22%, there should be that control+ligand.

-   b. Incubate the plates at 37° C. for 18 hours in a CO₂ incubator.-   c. Add 40 uL of lysis buffer (part of sICAM-1 kit from Endogen    EH-5400) to the wells and pipet up and down.-   d. Shake for M hour on rocker platform RT at 150 RPMs/min.-   e. Make a 1:1 dilution (40 uL of lysate+40 uL of diluent from    sICAM-1 kit from Endogen EH-5400) in Falcon microtest flexible assay    plates.-   f. Pipet 25 uL of this mixture into ELISA kit plate (sICAM-1    precoated plate, Endogen) Also, pipet 25 uL of standards on the    plate.-   g. Add 75 uL of HRP- conjugated Anti-ICAM-1 antibody (from sICAM-1    kit from Endogen EH-5400) to all wells except the blank.-   h. Cover the plate and incubate 2 hrs at RT on shaking platform 150    RPM/min.-   i. Wash plate using T-Cell protocol on plate washer. The wash buffer    is PBS+0.05% tween-20.-   j. Dissolve 1 tablet of OPD per 5 mL of substrate buffer (from    sICAM-1 kit from Endogen EH-5400, use 15 mL/plate).-   k. Pipet 100 uL into each well.-   l. Incubate uncovered for 30 minutes at RT.-   m. Add 50 uL of 2N H₂SO₄ to stop the, reaction.-   n. Read absorbance at 490 nM.-   Positive control: Cells Plus ligand-   Negative control: Cells only, no ligand-   Experimental control: Cells plus ligand plus 10 ug of 5c8 mAb.    Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativeof, rather than limiting on, the invention disclosed herein. All changeswhich come within the meaning and range of equivalency of the claims areintended to be embraced therein.

1. A compound, which interrupts CD40:CD154 interaction, of the formula:

Formula I, wherein, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independentlyselected from the group consisting of H, C1-5 alkyl, C1-5 alkyl-aryl,C1-5 alkyl-cycloalkyl, C1-5 alkyl-heteroaryl, C1-5 alkenyl-heterocyclo,cycloalkyl, cycloalkyl-aryl, C1-5 alkenyl-aryl, CR^(g)R^(h)CO₂H andCR^(g)R^(h)CO₂alkyl; wherein aryl or heteroaryl are optionallysubstituted with one to four substituents selected from R^(d) and alkylis optionally substituted with R^(i); or R¹ and R², R³ and R⁴, R⁵ andR⁶, and R⁷ and R⁸ independently form, together with the nitrogen towhich they are attached, a 5-6 membered ring which is optionallysubstituted with a heteroatom selected from the group consisting ofnitrogen, sulfur and oxygen, wherein the nitrogen may optionally besubstituted with alkyl or alkyl-aryl and the ring may optionally besubstituted with R^(j); R^(a), R^(b) and R^(c) are independentlyselected from the group consisting of H, alkyl and alkyl-aryl; R^(d) isselected from the group consisting of halogen, alkyloxy, NO₂, NH₂,alkyl, SO₂NH₂, hydroxyl and aryl; R^(e) and R^(f) are independentlyselected from the group consisting of H, alkyl and cycloalkyl; orR^(e)R^(f) together with the atom to which they are attached form a 4-7membered ring containing zero to three heteroatoms selected from thegroup consisting of nitrogen, oxygen and sulfur, wherein R^(e)R^(f)together with the atom to which they are attached may be fused to one ortwo aromatic rings; R^(g) and R^(h) are independently selected from thegroup consisting of H, alkyl, aryl, cycloalkyl, alkyl-aryl andalkyl-cycloalkyl; wherein alkyl is optionally substituted with R^(k);and aryl is optionally substituted with R^(l); or R^(g)R^(h) togetherwith the carbon to which they are attached form a 5-8 membered ringwhich may be bicyclic; R^(i) is selected from the group consisting of:NR^(e)R^(f), alkyloxy, aryloxy, C(O)aryl, aryl, OC(O)alkyl, alkylaryloxyand hydroxyl; wherein alkyl is optionally substituted with halogen andaryl is optionally substituted with R^(d); R^(j) is selected from thegroup consisting of (CH₂)_(y)NR^(e)R^(f), (CH₂)_(z)OH, CO₂H,C(O)N(alkyl)₂, C(O)NH₂, alkylaryl and aryl; wherein aryl is optionallysubstituted with one to four substituents selected from R^(d); R^(k) isselected from the group consisting of C(O)NH₂, C(O)OH, alkylthio, NH₂,heteroaryl, heteroalkyl, NHC(NH)NH₂, C1-5 alkylhydroxyl, hydroxyl andalkyloxy; R^(l) is selected from the group consisting of hydroxyl, NO₂and C1-5 alkyl; U is selected from the group consisting of O, S(O)_(y),NR^(c), NR^(c)C(O), NR^(c)C(O)NR^(c); W is selected from the groupconsisting of (CR^(a)R^(b))_(x), (CR^(a)R^(b))_(y)U(CR^(a)R^(b))_(y),C2-3 alkynyl, C2-3 alkenyl; each X is independently 0-3; each Y isindependently 0-2; each Z is independently 1-2.
 2. A compound, whichinterrupts CD40:CD154 interaction, of the formula:

R⁹ is selected from the group consisting of C1-5 alkyl-aryl, cycloalkyland alkenylaryl; wherein the alkyl is optionally substituted with aryland the aryl is optionally substituted with one to four halogens, aryl,NH₂ or NO₂; R¹⁰ is selected from the group consisting of alkyl andcycloalkyl; and R¹¹ is selected from the group consisting of H andalkyl.
 3. A compound, which interrupts CD40:CD154 interaction, of theformula:

wherein, R⁹ is selected from the group consisting of C1-5 alkyl-aryl;wherein alkyl and aryl are optionally substituted with aryl.
 4. Thecompound according to claim 1, wherein said compound is derived fromS-amines and wherein said compound is selected from the group consistingof:


5. The compound according to claim 1 or 2, wherein said compound isderived from S-amines and wherein said compound is selected from thegroup consisting of:


6. The compound according to any one of claims 1, 2 or 3, wherein saidcompound is derived from S-amines and wherein said compound is selectedfrom the group consisting of:


7. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound according to any one of claims 1-6.
 8. A methodfor attenuating severity of a condition associated with inappropriateCD154 mediated activation in a subject, comprising the step ofadministering an effective amount of a pharmaceutical compositionaccording to claim 7 to the subject.
 9. A method for suppressing effectsof a condition associated with inappropriate CD154 mediated activationin a subject, comprising the step of administering an effective amountof a pharmaceutical composition according to claim 7 to the subject. 10.A method for preventing development of a condition associated withinappropriate CD154 mediated activation in a subject, comprising thestep of administering an effective amount of a pharmaceuticalcomposition according to claim 7 to the subject.
 11. A method fordelaying onset of a condition associated with inappropriate CD154mediated activation in a subject, comprising the step of administeringan effective amount of a pharmaceutical composition according to claim 7to the subject.
 12. A method for inhibiting a condition associated withinappropriate CD154 mediated activation in a subject, comprising thestep of administering an effective amount of a pharmaceuticalcomposition according to claim 7 to the subject.
 13. A method forreversing a condition associated with inappropriate CD154 mediatedactivation in a subject, comprising the step of administering aneffective amount of a pharmaceutical composition according to claim 7 tothe subject.
 14. A method for treating a condition associated withinappropriate CD154 mediated activation in a subject, comprising thestep of administering an effective amount of a pharmaceuticalcomposition according to claim 7 to the subject.
 15. A method forpreventing a condition associated with inappropriate CD154 mediatedactivation in a subject, comprising the step of administering aneffective amount of a pharmaceutical composition according to claim 7 tothe subject.
 16. The method according to any one of claims 8-15, whereinthe subject is a primate.
 17. The method according claim 16, whereinsaid primate is a human.
 18. The method according to any one of claims8-15, wherein the condition is an unwanted immune response.
 19. Themethod according to any one of claims 8-15, wherein the condition is anunwanted inflammatory response.
 20. The method according to any one ofclaims 8-15, wherein the condition is an autoimmune disease.
 21. Themethod according to any one of claims 8-15, wherein the condition is anallergy.
 22. The method according to any one of claims 8-15, wherein thecondition is an inhibitor response to a therapeutic agent.
 23. Themethod according to any one of claims 8-15, wherein the condition isrejection of a donor organ.
 24. The method according to any one ofclaims 8-15, wherein the condition is a B cell cancer.
 25. The methodaccording to any one of claims 8-15, wherein the condition is selectedfrom the group consisting of: systemic lupus erythematosis, lupusnephritis, lupus neuritis, asthma, chronic obstructive pulmonarydisease, bronchitis, emphysema, multiple sclerosis, uveitis, Alzheimer'sdisease, traumatic spinal cord injury, stroke, atherosclerosis, coronaryrestenosis, ischemic congestive heart failure, cirrhosis, hepatitis C,diabetic nephropathy, glomerulonephritis, osteoarthritis, rheumatoidarthritis, psoriasis, atopic dermatitis, systemic sclerosis,radiation-induced fibrosis, Crohn's disease, ulcerative colitis,multiple myeloma and cachexia.
 26. A compound, which interruptsCD40:CD154 interaction, of the formula: